PROJECT SUMMARY Histone methyltransferases (HMTs) are a group of enzymes that represent a critical focal point in the linkage of cellular signals to the regulation of gene expression. These proteins are not only strongly associated with numerous diseases (e.g. cancers, neurodegenerative diseases, and autoimmune disorders) but also have properties that make them excellent targets for drugs. Thus, HMTs provide an exceptional therapeutic opportunity. Currently, the tests that are used to discover new HMT drugs rely primarily on reagents known as peptides. These peptides represent only a tiny portion of nucleosomes, the natural HMT substrate. The use of the peptides in tests for HMT activity poorly mimics the physical interactions that occur between HMTs and nucleosomes in a living cell. This can result in findings that are often not recapitulated in living organisms. EpiCypher has developed the ability the manufacture and commercialize pure recombinant nucleosomes, known as dNucs, that are highly similar to naturally occurring nucleosomes. Here, we propose to develop the reagents and methods needed to manufacture and distribute HMT activity tests that utilize dNucs as key components. These kits will allow researchers to efficiently screen up to tens of thousands of potential drug molecules for HMT modulating activity. During the Phase I portion of this work, we developed the AlphaNuc? HMT assay, a novel nucleosome-based HMT activity test using PerkinElmer's AlphaScreen technology. This study provided strong proof-of-concept that EpiCypher's dNuc technology will provide the resolution and reproducibility needed for rapidly testing potential drug molecules. During this Phase II work, EpiCypher will expand the HMT AlphaNuc? assay for a family of HMTs with known relevance in disease, which includes the high sought after drug target, the NSD2 enzyme. This work will include an optimization strategy that ensures that the tests are robust and suitable for the high-throughput screening of potential drug molecules. As part of this development process, and to check the dNuc reagents and protocols in a real world scenario, EpiCypher will conduct a pilot screen of a chemical library for inhibitors of the NSD2 HMT using dNucs as substrates. This screen will serve to validate this platform for use in drug screens and to provide insight into how changes in dNuc methylation status alter screening outcomes. Finally, we will develop the methods needed to scale up commercial manufacturing of dNucs. These optimizations will enable EpiCypher to generate large quantities of dNucs ?to order? and at a dramatically reduced price point.